The expression of EPHB4 was detected via phycoerythrin (PE)‐conjugated EPHB4 antibody (R&D Systems, Inc., Minneapolis, MN, USA) staining. For the detection of EPHB4‐CAR expression, transduced T cells were stained using goat anti‐Ephrin B2 antibody (R&D Systems) and then stained using PE‐conjugated anti‐goat IgG antibody (R&D Systems). Allophycocyanin (APC)‐conjugated anti‐CD3 antibody, APC‐conjugated anti‐CD8a antibody, fluorescein isothiocyanate (FITC)‐conjugated CD4 antibody, FITC‐conjugated anti‐CD45RA antibody, and APC‐conjugated anti‐CCR7 antibody (all from BioLegend) were used for the characterisation of the CAR‐T cell phenotype. To determine the binding capacity of human Ephrin B2 to cynomolgus EPHB4, 293‐human EPHB4‐GFP and 293‐cynoEPHB4‐GFP cells were incubated with the recombinant human Ephrin B2‐Fc Chimera Protein (R&D Systems) for 20 min on ice and then stained using APC‐conjugated anti‐human IgG‐Fc antibody (BioLegend). Detailed antibody information is presented in Supplementary table 2 . All flow cytometry data were acquired using BD FACS Accuri C6 Plus (BD Biosciences) and analysed using the FlowJo Software (Tree Star, Inc., Ashland, OR, USA).
Apc conjugated anti human igg fc antibody
Manufactured by BioLegend
Sourced in United States
The APC-conjugated anti-human IgG-Fc antibody is a laboratory reagent used for the detection and quantification of human IgG-Fc in various immunoassays. It is a monoclonal antibody that is conjugated to the fluorescent dye Allophycocyanin (APC), which allows for sensitive detection of the target IgG-Fc.
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Lab products found in correlation
Zeocin, by Thermo Fisher Scientific (2 mentions)
Facs accuri c6 plus, by BD (1 mentions)
Apc conjugated anti cd3 antibody, by BioLegend (1 mentions)
Apc conjugated anti cd8a antibody, by BioLegend (1 mentions)
Apc conjugated anti ccr7 antibody, by BioLegend (1 mentions)
Recombinant human ephrin b2 fc chimera protein, by R&D Systems (1 mentions)
Fitc conjugated cd4 antibody, by BioLegend (1 mentions)
Cytexpert software, by Beckman Coulter (1 mentions)
Cytoflex cytometer, by Beckman Coulter (1 mentions)
Facscalibur, by BD (1 mentions)
Facsymphony a5 analyser, by BD (1 mentions)
5 protocols using apc conjugated anti human igg fc antibody
1
EPHB4 Expression and Binding Assay
2
Jurkat Cell CD3 and EGFRvIII-BsAb Binding
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Jurkat cells were resuspended in FACS buffer on ice and incubated with the CD3 mAb or the EGFRvIII-BsAb at a series of concentrations, followed by APC-conjugated antihuman IgG-Fc antibody (409305; Biolegend, San Diego, CA, USA). Samples were analyzed with a CytoFLEX cytometer using CytExpert software (BECKMAN COULTER, Brea, CA, USA).
3
Establishing MERS-S Expressing 293T Cell Line
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The 293T cells (ATCC, CRL-3216) were transfected with the pcDNA4-MERS-S expression vector using the Lipofectamine transfection system (Invitrogen), followed by zeocin selection (500 μg/mL) 24 h post-transfection. After recovery, the stable pool was subcloned by limited dilution, and the clones with high expression of MERS-S were identified by flow cytometry. For the flow cytometry assay, the cells were incubated with h2E6 anti-MERS antibody against the cell-surface antigen for 30 min at 4 °C and the unconjugated antibodies were washed out with FACS washing buffer (2% FBS in PBS), followed by another 30 min incubation with APC-conjugated anti-human IgG Fc antibody (Biolegend) and washing procedure. Isotype antibodies were included, substituting the anti-MERS antibody as negative controls. The luminescence intensity of the stained cells was analyzed by flow cytometry (FACS Calibur, BD Biosciences). The clones highly expressed the MERS antigen, which were identified by flow cytometry, were further selected by the ADCC reporter gene assay (RGA). The selected 293T/MERS cell line was cultured in high glucose DMEM medium with 10% fetal bovine serum and 500 μg/mL zeocin (Gibco).
4
Characterization of SARS-CoV-2 Spike Protein Interactions
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Cells were resuspended in staining buffer (PBS + 2% FBS) and incubated with fluorochrome‐conjugated antibodies for 30 min at 4°C. CD8 T cells were identified with CD8‐Pacific Blue antibody (Biolegend). Activation of CD8 CAR‐T cells was determined with CD25 staining using the CD25‐APC antibody (Biolegend). CAR expressions of ACE2 CAR and anti‐Spike CAR and ACE2 expression of ACE2‐293 cells were determined with SARS‐CoV‐2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) incubation followed with APC Goat anti‐mouse IgG2a Fc Antibody (Invitrogen) staining and RFP expression. CAR expression of anti‐CD19 CAR was determined with Human CD19 (20–291) Protein, Fc Tag, low endotoxin (Super affinity) (Acro) followed by a secondary staining with APC conjugated anti‐human IgG Fc Antibody (Biolegend) and RFP expression. For cytotoxicity assay analysis, stably Spike protein‐expressing T2 and 293 cell lines were identified with GFP marker. For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2/ACEH Protein, Fc, Avitag (Acro Biosystems) and then stained with APC anti‐human IgG Fc Antibody clone HP6017 (Biolegend). Samples were acquired on a BD FACSymphony A5 analyser, and data were analysed using FlowJo (BD Biosciences).
5
CD22-TCB Cell Binding Assay
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Cell binding assay was performed to evaluate the bioactivity of six CD22-TCBs by using CD22+ NALM-6G cell line. In brief, NALM-6G cells were harvested in logarithmic phase with 5E5/well, and incubated with 5 μg CD22-TCB on ice for 30 min. After incubation, samples were incubated with secondary antibody (APC conjugated anti-human IgG Fc Antibody, Biolegend™) under the same condition. Finally, samples were analyzed by CytoFLEX cytometry.
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